Project Summary Clostridium difficile, the most common cause of hospital-acquired infectious diarrhea, is responsible for 250,000 infections and 14,000 deaths each year in the United States alone. Much of the pathology associated with C. difficile infections is caused by two major toxins, TcdA and TcdB. Multiple studies have demonstrated the critical role TcdA and TcdB play in causing C. difficile infections. Previous studies have established that expression of the toxins is induced in response to nutrient limitation. Moreover, several factors that mediate this response have been identified, including an alternative sigma factor (TcdR), a global regulator that responds to GTP and branched chain amino acids (CodY), and a global regulator that responds to easily metabolized carbon sources like glucose (CcpA). Here we describe the use of single cell analysis to monitor expression of the toxin genes in C. difficile. We fused the gene for a red fluorescent protein (RFP) to the promoter for toxin A, which allowed us to monitor toxin expres- sion in single cells by fluorescence microscopy and flow cytometry. Surprisingly, tcdA-rfp expression appears to be ?bistable.? In stationary phase, when toxin expression is highest, about 30% of the cells are ?TcdA-ON? and the mean fluorescence intensity of these cells is ~50 fold higher than in the ?TcdA- OFF? population. We find that bistability is dependent upon the levels of TcdR. These findings raise several important questions: How is bistability controlled? Are other genes regulated in a bistable man- ner? What is the biological significance of this phenotypic diversity? Here we propose to address the first two questions by (a) testing the hypothesis that bistable expression of toxin production is mediated by global regulators of gene expression controlling levels of TcdR and (b) identifying genes co-regulated with tcdA. Achieving the aims of this proposal will lead to a better understanding of the mechanisms by which toxin gene expression is controlled in C. difficile at the level of the single cell.